Method for differentiating stromal or peri-cells

Abstract

The present disclosure relates to methods of differentiating pluripotent stem cells into stromal cells or peri-cells. The present disclosure also relates to stromal cells or peri-cells prepared by such methods, organoids comprising such stromal cells or peri-cells, and methods of use thereof. The present disclosure also relates to differential media for use therein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 390,458, filed on July 19, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure relates to methods for differentiating pluripotent stem cells into stromal cells or pericytes. The present disclosure also relates to stromal cells or pericytes prepared by such methods, organoids containing such stromal cells or pericytes, and methods of using the same. The present disclosure also relates to differentiation medium for use in the above. Background Art

[0003] Pluripotent stem cells (PSCs) are undifferentiated or partially differentiated cells that can differentiate into a variety of other cell types. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells derived from adult somatic cells that are genetically reprogrammed to an embryonic stem cell (ESC)-like state by expressing genes and factors that are important for maintaining the most typical characteristics of embryonic stem cells (ESCs). iPSCs have recently attracted attention in the medical community because they solve many of the obstacles associated with the use of embryonic stem cells and can generate patient-specific PSCs that can be genetically corrected, differentiated into adult cell lineages, and returned to the same patient as an autologous transplant. Yamanaka et al., Cell Stem Cell. 1(1): 39-49 (2007); Nishikawa et al., Nat. Rev. Mol. Cell Biol. 9: 725 (2008). In addition to being used for genetic diseases, iPSCs can also be used for tissue regeneration and disease modeling. Kogut et al., Methods Mol. Biol. 1195: 1-12 (2014). PSCs and iPSCs can be differentiated into many different cell types, including endothelial cells (EC). Jang et al., Am. J. Pathol. 189(3): 502-512 (2019); Gu et al., Curr. Protoc. Hum. Genet. published online 2018 Jul. 6. doi: 10.1002 / cphg.64. Summary of the invention

[0004] The present disclosure provides methods for differentiating pluripotent stem cells (PSCs) into stromal cells or pericytes.

[0005] In some aspects, the method comprises (i) culturing pluripotent stem cells (e.g., iPSCs) in a basal medium comprising bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), a glycogen synthase kinase 3 (GSK3) inhibitor, activin A, and a Rho-associated coiled-coil kinase (ROCK) inhibitor; (ii) culturing the cells in (i) in a basal medium comprising VEGF and a transforming growth factor β (TGFβ) inhibitor; and (iii) culturing the cells in (ii) in a basal medium comprising TGFβ3 and platelet-derived growth factor (PDGF) to form stromal cells or pericytes.

[0006] In some aspects, the method further comprises, prior to (iii), culturing the cells in (ii) in a basal medium.

[0007] In some aspects, the method further comprises (iv) culturing the cells in (iii) in a basal medium.

[0008] In some aspects, the cells in (ii) are not passaged prior to (iii).

[0009] In some aspects, the method comprises: (i) culturing PSCs (e.g., iPSCs) in a basal medium comprising BMP4, VEGF, a GSK3 inhibitor, and activin A; (ii) culturing the cells in (i) in a basal medium comprising VEGF and a TGFβ inhibitor; (iii) culturing the cells in (ii) in a basal medium; and (iv) culturing the cells in (iii) in a basal medium comprising TGFβ3 and PDGF to form stromal cells.

[0010] In some aspects, the method further comprises (v) culturing the cells in (iv) in a basal medium.

[0011] In some aspects, the cells in (ii) are not passaged prior to (iii).

[0012] In some aspects, the culture medium in (i) further comprises a ROCK inhibitor. In some aspects, the ROCK inhibitor is Y-27632. In some aspects, Y-27632 is present in the culture medium at a concentration of about 10 μM.

[0013] In some aspects, the GSK3 inhibitor is CHIR99021. In some aspects, CHIR99021 is present in the culture medium at a concentration of about 1.5 μM.

[0014] In some aspects, BMP4 is present in the culture medium at a concentration of about 30 ng / mL.

[0015] In some aspects, VEGF is present in the culture medium at a concentration of about 50 ng / mL.

[0016] In some aspects, activin A is present in the culture medium at a concentration of about 25 ng / mL.

[0017] In some aspects, the TGFβ inhibitor is SB 431542. In some aspects, SB 431542 is present in the culture medium at a concentration of about 10 μM.

[0018] In some aspects, TGFβ3 is present in the culture medium at a concentration of about 2 ng / mL.

[0019] In some aspects, the PDGF is PDGF-BB. In some aspects, PDGF-BB is present in the culture medium at a concentration of about 4 ng / mL.

[0020] In some aspects, the stromal cells are pericytes.

[0021] In some aspects, at the time of initiating the culturing of (i), the iPSCs are not fully confluent.

[0022] In some aspects, at the time of initiating the culturing of (i), the iPSCs are at about 50% to about 80% confluence.

[0023] The present disclosure also provides stromal cells prepared by the differentiation methods disclosed herein, pericytes prepared by the differentiation methods disclosed herein, organoids comprising the stromal cells disclosed herein, organoids comprising the pericytes disclosed herein, and certain methods of use thereof.

[0024] The present disclosure also provides certain differentiation medium. In some aspects, the differentiation medium comprises a basal medium, BMP4, VEGF, a GSK3 inhibitor, activin A and a ROCK inhibitor. In some aspects, the differentiation medium comprises a basal medium, about 30 ng / mL BMP4, about 50 ng / mL VEGF, about 1.5 μM CHIR99021, about 25 ng / mL activin A and about 10 μM Y-27632. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Some aspects of the present invention are described herein by way of example only and with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is emphasized that the details shown are by way of example only and for the purpose of illustrative discussion of various aspects of the present invention.

[0026] Figure 1A is an exemplary whole-cell image of day 1 of the differentiation protocol described in Example 1.

[0027] Figure 1B is an exemplary whole-cell image of day 2 of the differentiation protocol described in Example 1.

[0028] Figure 1C is an exemplary whole-cell image of day 4 of the differentiation protocol described in Example 1.

[0029] Figure 1D is an exemplary whole-cell image of day 5 of the differentiation protocol described in Example 1.

[0030] Figure 1E is an exemplary whole-cell image of day 7 of the differentiation protocol described in Example 1.

[0031] Figure 1F is an exemplary whole-cell image of day 9 of the differentiation protocol described in Example 1.

[0032] Figure 1G is an exemplary whole-cell image of day 10 of the differentiation protocol described in Example 1.

[0033] Figure 1H is an exemplary whole-cell image of day 17 of the differentiation protocol described in Example 1.

[0034] Fig. 1I -IJ is an exemplary whole-cell image of the final test differentiated cells described in Example 1.

[0035] Figure 2 The results of tube formation of the test differentiated cells of Example 1 co-cultured with human pulmonary artery endothelial cells (HPAEC) treated or not with the angiogenesis inhibitor imatinib mesylate are shown.

[0036] Figures 3A-3C shows that at the three hour time point, HPAECs were co-cultured with the test differentiated cells of Example 1 ( Figure 3A ), HPAEC co-cultured with test differentiated cells treated with imatinib mesylate ( Figure 3B ) and co-culture of HPAEC and placental pericytes ( Figure 3C )'s exemplary image of tube formation. DETAILED DESCRIPTION I. General Definitions

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application (including definitions therein) shall prevail. Unless otherwise required by the context, terms in the singular shall include the plural form, and terms in the plural form shall include the singular form. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes, just as each individual publication or patent application is expressly and individually indicated to be incorporated by reference.

[0038] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. These materials, methods and examples are for illustrative purposes only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0039] In order to further clarify the present disclosure, the following terms and definitions are provided.

[0040] The singular forms "a" and "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In some aspects, the terms "a" or "an" mean "single". In other aspects, the terms "a" or "an" include "two or more" or "plurality".

[0041] The term "about" as used herein means approximately, roughly, approximately, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values. Generally, the term "about" is used herein to modify a numerical value by floating it within a range of 10% (higher or lower) above or below a numerical value.

[0042] Throughout the disclosure of the present invention, various aspects of the present invention are presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity and should not be interpreted as an unchangeable limitation on the scope of the present invention. Therefore, the description of a range should be deemed to have specifically disclosed all possible sub-ranges and single values ​​within the range. For example, a description such as a range of 1 to 6 should be deemed to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and single numbers within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the range, this rule applies. The listed numerical ranges include the numbers that define the range and cover each integer within the defined range.

[0043] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers that define the range. When a numerical range is listed, it should be understood that each intermediate integer value between the upper and lower limits listed in the range and each fractional value thereof, as well as each subrange between these values, is also specifically disclosed. The upper and lower limits of any range may be independently included in the range or excluded from the range, and any range that includes the upper limit, the lower limit, both or neither is also included in the disclosure of the present invention. Therefore, the ranges listed herein should be understood to be a shorthand representation of all numerical values ​​in the range, including the endpoint values ​​listed. For example, a range of 1 to 10 should be understood to include any number, combination of numbers or subrange in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0044] When a numerical value is explicitly listed, it should be understood that the numerical value of the quantity or amount approximately the same as the listed numerical value is also within the scope of the present disclosure. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. On the contrary, when different elements or groups of elements are disclosed separately, their combinations are also disclosed. When any element of the invention disclosure is disclosed as having multiple alternatives, examples of the invention disclosure in which each alternative is excluded individually or in any combination with other alternatives are also disclosed; more than one element of the invention disclosure can have such exclusions, and all combinations of elements with such exclusions are also disclosed herein.

[0045] As used herein, the term "and / or" should be considered as a specific disclosure of the presence or absence of the other of the two specified features or components. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0046] It should be understood that wherever herein aspects are described with the language "comprising," similar aspects described with "consisting of" and / or "consisting essentially of" are also provided. II. Differentiation Methods

[0047] The present disclosure relates to improved methods for differentiating pluripotent stem cells (PSCs) into stromal cells or pericytes. Such methods result in, for example, improved cell viability, yield and / or properties of the differentiated cells.

[0048] As used herein, the term "differentiation" and "differentiating" refers to the process of inducing or reprogramming young or immature cells (e.g., pluripotent stem cells) into more mature or specialized cells (e.g., stromal cells or pericytes). In general, differentiation of pluripotent stem cells can be achieved, for example, by changing the culture conditions of the cells (such as changing the stimulator in the culture medium or the physical state of the cells).

[0049] As used herein, the terms "pluripotent stem cell," "pluripoent stem cells," and "PSC" refer to young or immature cells that can develop into more mature or specialized cells, such as stromal cells or pericytes.

[0050] In some aspects, PSCs include, but are not limited to, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells, adult stem cells, or combinations thereof. In some aspects, PSCs are from humans. In some aspects, PSCs are from animals. In some aspects, the animals are sheep, pigs, or primates.

[0051] As used herein, the terms "induced pluripotent stem cell," "induced pluripoent stem cells" and "iPSC" refer to cells generated from differentiated adult, neonatal or fetal cells that have been induced or reprogrammed to become pluripotent stem cells.

[0052] As used herein, the term "stromal cells" or "SC" refers to cells that function as connective tissue cells of an organ, supporting the function of parenchymal cells of a particular organ. As used herein, stromal cells include mature stromal cells, stromal progenitor cells, and stromal precursor cells.

[0053] In some aspects, the stromal cells described herein are pericytes. As used herein, the term "pericyte" refers to fibroblast-like cells with extensive cytoplasmic processes that surround endothelial cells in arterioles, capillaries, and venules, covering 22% to 99% of the endothelial cell surface. Pericytes are embedded in the basement membrane of capillaries, where they communicate with endothelial cells by direct physical contact and paracrine signaling.

[0054] In some aspects, the differentiation methods provided herein include certain cell culture conditions, such as culturing cells in certain culture media.

[0055] As used herein, the terms "cell culture", "cell culturing", "culture", "culturing" and "cultured" refer to the maintenance, growth and / or differentiation of cells in an in vitro environment. The terms "cell culture medium", "cell culture media", "culture medium" and "culture media" refer to a composition for culturing cells that contains nutrients that maintain cell viability, support proliferation and, optionally, differentiation. A cell culture medium may contain one or more of the following: one or more salts, one or more buffers, one or more amino acids, glucose or other sugars, one or more antibiotics, serum or serum substitutes, and other components such as growth factors, vitamins, and the like.

[0056] In some aspects, the differentiation methods provided herein refer to the cell culture medium as a "basal medium" (sometimes referred to herein as a "differentiation medium" or "differentiation medium") supplemented with other components. As used herein, "basal culture medium" or "basal culture medium" refers to a composition containing the most basic elements required for the maintenance, growth and / or differentiation of cells in an in vitro environment. Examples of basal culture media include, but are not limited to, Dulbecco's modified Eagle's medium (DMEM), MEM, Iskoff's modified Dulbecco's medium (IMDM), Glasgow modified MEM (GMEM), DMEM / F12, Leibovitz L-15, RPMI-1640, CMRL, Ham F10, and Ham F12. In some aspects, the basal culture medium is supplemented with one or more other components, such as one or more amino acids, one or more antibiotics, serum, one or more growth factors. Such components are well known in the art and are further described herein.

[0057] In some aspects, the differentiation methods provided herein include certain cell culture conditions, such as passage of cells in certain culture media. As used herein, the terms "passage," "passaged," and "passaging" refer to the act of subdividing cells at lower concentrations and inoculating them into one or more cell culture surfaces or containers when the cells have proliferated to a desired degree. Passaging generally involves mechanical or enzymatic separation (e.g., at a certain cell density) prior to inoculation. Methods for passaging cells are well known and further described herein.

[0058] In some aspects, the culture and passage in the differentiation methods provided herein are carried out under the condition of coating one or more substrates on the cell culture surface or container. Such substrates include, but are not limited to, vitronectin, gelatin, laminin, fibronectin, collagen (e.g., collagen I, collagen IV or a combination thereof), elastin, osteopontin, thrombospondin, matrix mixtures produced by naturally occurring cell lines (such as Matrigel, TM ) and synthetic or artificial surfaces (e.g., polyamine monolayers and carboxyl-terminated monolayers) or combinations thereof. Methods for coating substrates onto cell culture surfaces or containers are well known and are further described herein.

[0059] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing bone morphogenetic protein 4 (BMP4). BMP4 stimulates differentiation of overlying ectodermal tissue and is known to stimulate bone formation in adult animals.

[0060] In some respects, BMP4 exists with the concentration of about 10ng / mL to about 50ng / mL (or any numerical value or numerical range) in basal medium, and the concentration for example includes about 20ng / mL to about 50ng / mL, about 30ng / mL to about 50ng / mL, about 40ng / mL to about 50ng / mL, about 10ng / mL to about 40ng / mL, about 20ng / mL to about 40ng / mL, about 30ng / mL to about 40ng / mL, about 10ng / mL to about 30ng / mL, about 20ng / mL to about 30ng / mL or about 10ng / mL to about 20ng / mL. In some respects, BMP4 exists with the concentration of about 10ng / mL, about 20ng / mL, about 30ng / mL, about 40ng / mL or about 50ng / mL in basal medium. In some respects, BMP4 exists with the concentration of about 30ng / mL in basal medium.

[0061] In other aspects, the basal medium in the differentiation method provided herein does not comprise BMP4 or is substantially free of BMP4. As used herein, the term "substantially free of" refers to at least 95% free of, 96% free of, 97% free of, 98% free of, 99% free of or 100% free of the culture medium of BMP4, or the culture medium with the BMP4 of undetectable amount, as known in the art and the method further described below measured. The term "does not comprise" refers to the culture medium without BMP4, or the culture medium with the BMP4 of undetectable amount, as known in the art and the method further described below measured.

[0062] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing vascular endothelial growth factor (VEGF). VEGF is a signaling protein that promotes the growth of new blood vessels. VEGF is part of the mechanism for restoring blood supply to cells and tissues when they are deprived of oxygen due to obstructed blood circulation.

[0063] In some aspects, VEGF is present in a concentration of about 10 ng / mL to about 100 ng / mL (or any value or range thereof) in a basal medium, for example, including about 25 ng / mL to about 100 ng / mL, about 50 ng / mL to about 100 ng / mL, about 75 ng / mL to about 100 ng / mL, about 10 ng / mL to about 75 ng / mL, about 25 ng / mL to about 75 ng / mL, about 50 ng / mL to about 75 ng / mL, about 10 ng / mL to about 50 ng / mL, about 25 ng / mL to about 50 ng / mL or about 10 ng / mL to about 25 ng / mL. In some aspects, VEGF is present in a concentration of 10 ng / mL, about 25 ng / mL, about 50 ng / mL, about 75 ng / mL or about 100 ng / mL in a basal medium. In some aspects, VEGF is present in a concentration of about 50 ng / mL in a basal medium.

[0064] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing a glycogen synthase kinase 3 (GSK3) inhibitor. GSK3 is a serine / threonine protein kinase that mediates the addition of phosphate molecules to certain serine and threonine amino acids of cell substrates (e.g., glycogen synthase). This phosphorylation typically results in inhibition of the substrate. GSK3 is also involved in controlling the response of cells to damaged DNA, Wnt signaling, and the phosphorylation of Ci in the Hedgehog (Hh) pathway, targeting it for proteolysis to an inactive form.

[0065] As used herein, "GSK3 inhibitor" refers to a compound that inhibits one or more GSK3 enzymes. The GSK3 enzyme family is well known, and many variants have been described (e.g., Schaffer et al., Gene, 302: 73-81, 2003). Specific examples of GSK3 inhibitors include, but are not limited to, Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418, CT99021, CT20026, SB415286, SB216763, AR-A014418, lithium, SB415286, and TDZD-8. Other exemplary GSK3 inhibitors include, but are not limited to, BIO(2'Z,3'E)-6-bromoindirubin-3'-oxime (GSK3 inhibitor IX); BIO-acetyloxime (2'Z,3'E)-6-bromoindirubin-3'-acetyloxime (GSK3 inhibitor X); (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3 inhibitor XIII); pyridocarbazole-cyclopentadienylruthenium complex (GSK3 inhibitor XV); TDZD-8,4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3β inhibitor I); 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3β inhibitor II); OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3β inhibitor III); α-4-dibromoacetophenone (GSK3β inhibitor VII); AR-AO14418 N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β inhibitor VIII); 3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK3β inhibitor XI); TWS119-pyrrolopyrimidine compound (GSK3β inhibitor XII); L803 H-KEAPP APPQSpP-NH2 or its myristoylated form (GSK3β inhibitor XIII); 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β inhibitor VI); AR-AO144-18; SB216763; and SB415286. In some aspects, the GSK3 inhibitor is CHIR99021.

[0066] In some aspects, GSK3 inhibitor (for example, CHIR99021) is present in a concentration of about 0.5 μM to about 3 μM (or any value or range thereof) in a basal medium, and the concentration, for example, includes about 0.5 μM to about 1.5 μM, about 0.5 μM to about 1 μM, about 0.5 μM to about 1.5 μM, about 0.5 μM to about 1 μM, or about 0.5 μM to about 1 μM. In some aspects, GSK3 inhibitor (for example, CHIR99021) is about 0.5 μM, about 1 μM, about 1.5 μM or about 3 μM. In some aspects, GSK3 inhibitor (for example, CHIR99021) is about 1.5 μM.

[0067] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing activin A. Activin A is a dimeric glycoprotein that belongs to the transforming growth factor β (TGFβ) family. It regulates a variety of biological functions, including hormone homeostasis, gonadal function, muscle growth, immunity, inflammation, and bone remodeling.

[0068] In some aspects, activin A exists in basal medium with a concentration of about 5ng / mL to about 50ng / mL (or any numerical value or numerical range thereof), and the concentration for example includes about 10ng / mL to about 50ng / mL, about 25ng / mL to about 50ng / mL, about 5ng / mL to about 25ng / mL, about 10ng / mL to about 25ng / mL, or about 5ng / mL to about 10ng / mL. In some aspects, activin A exists in basal medium with a concentration of about 5ng / mL, about 10ng / mL, about 25ng / mL or about 50ng / mL. In some aspects, activin A exists in basal medium with a concentration of about 25ng / mL.

[0069] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing a Rho-associated kinase (ROCK) inhibitor. ROCK is a serine / threonine kinase that functions as a downstream effector of Rho kinase, wherein Rho kinase has three subtypes (RhoA, RhoB, and RhoC). A "ROCK inhibitor" can, for example, reduce ROCK expression and / or ROCK activity. Examples of ROCK inhibitors include, but are not limited to, polynucleotides, polypeptides, and small molecules. More specific examples of ROCK inhibitors include, but are not limited to, anti-ROCK antibodies, dominant negative ROCK variants, siRNA, shRNA, miRNA, and antisense nucleic acids targeting ROCK. Other examples of ROCK inhibitors include, but are not limited to, thiazovivin, Y-27632, Fasudil, AR122-86, Y-30141, WF-536, HA-1077, hydroxy-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N′-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxylic acid amide, and ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety. In some aspects, the ROCK inhibitor is Y-27632.

[0070] In some aspects, ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 1 μM to about 20 μM (or any value or range thereof) in a basal medium, for example, including about 1 μM to about 15 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, about 5 μM to about 20 μM, about 5 μM to about 15 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 10 μM to about 15 μM or about 15 μM to about 20 μM. In some aspects, ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 1 μM, about 5 μM, about 10 μM, about 15 μM or about 20 μM in a basal medium. In some aspects, ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 10 μM in a basal medium.

[0071] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium comprising a TGFβ inhibitor. TGFβ is a highly pleiotropic cytokine that plays an important role in wound healing, angiogenesis, immunomodulation, and cancer. As used herein, the term "TGFβ inhibitor" includes, but is not limited to, general TGF signaling inhibitors, or inhibitors specific for TGFβ receptors (e.g., ALK5), which may include antibodies for TGFβ receptors, dominant negative variants for TGFβ receptors, and siRNA and antisense nucleic acids that inhibit TGFβ receptor expression. Examples of TGFβ inhibitors include, but are not limited to, SB431542, A-83-01 (also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolyl)-1H-pyrazole-1-methanethioamide), 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO, BMP4, GW788388 (-4-[3-(pyridin-2-yl)-1H-pyrazole-4 -yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), SMI6, 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide, GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl SU5416, lerdelimumab (CAT-152), metelimumab (CAT-192), GC-1008, ID11, AP-12009, AP-11014, LY550410, LY580276, LY364947, LY2109761, and SB-431542 ; SD-208; SM16; NPC-30345; KI26894; SB-203580; SD-093; ALX-270-448; EW-7195; SB-525334; IN-1233; SKI2162; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (Morin); Activin-M108A; P144; soluble TBR2-Fc and pyrimidine derivatives and indolinone compounds reported in Roth et al., 2010. In some aspects, the TGFβ inhibitor is SB431542.

[0072] In some aspects, TGFβ inhibitor (e.g., SB431542) is present in a concentration of about 1 μM to about 20 μM (or any value or range thereof) in a basal medium, for example, including about 5 μM to about 20 μM, about 10 μM to about 20 μM, about 1 μM to about 10 μM, and about 1 μM to about 5 μM. In some aspects, TGFβ inhibitor (e.g., SB431542) is present in a concentration of about 1 μM, about 5 μM, about 10 μM, or about 20 μM in a basal medium. In some aspects, TGFβ inhibitor (e.g., SB431542) is present in a concentration of about 10 μM in a basal medium.

[0073] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing transforming growth factor β3 (TGFβ3). TGFβ3 is a cytokine involved in cell differentiation, embryogenesis and development. TGFβ3 is believed to regulate a molecule involved in cell adhesion and extracellular matrix (ECM) formation during palate development.

[0074] In some aspects, TGFβ3 exists in the basal medium with a concentration of about 0.5ng / mL to about 5ng / mL (or any numerical value or numerical range thereof), and the concentration, for example, includes about 1ng / mL to about 5ng / mL, about 2ng / mL to about 5ng / mL, about 0.5ng / mL to about 2ng / mL, about 1ng / mL to about 2ng / mL, or about 0.5ng / mL to about 1ng / mL. In some aspects, TGFβ3 exists in the basal medium with a concentration of about 0.5ng / mL, about 1ng / mL, about 2ng / mL or about 5ng / mL. In some aspects, TGFβ3 exists in the basal medium with a concentration of about 2ng / mL.

[0075] Some aspects of the differentiation method provided herein are included in culturing cells in a basal medium containing platelet-derived growth factor (PDGF). PDGF is a growth factor that regulates cell growth and division, plays an important role in angiogenesis, blood vessel growth from existing vascular tissue, mitosis, and the chemotaxis of mesenchymal cells. PDGF is a dimeric glycoprotein that can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or each subunit (PDGF-AB). In some respects, the PDGF is PDGF-BB.

[0076] In some aspects, PDGF (e.g., PDGF-BB) is present in a concentration of about 2ng / mL to about 8ng / mL (or any numerical value or numerical range thereof) in a basal medium, and the concentration, for example, includes 4ng / mL to about 8ng / mL, about 5ng / mL to about 8ng / mL, about 2ng / mL to about 5ng / mL, about 4ng / mL to about 5ng / mL, or about 2ng / mL to about 4ng / mL. In some aspects, PDGF (e.g., PDGF-BB) is about 2ng / mL, about 4ng / mL, about 5ng / mL, or about 8ng / mL. In some aspects, PDGF (e.g., PDGF-BB) is about 4ng / mL.

[0077] In some aspects, the differentiation method provided herein includes culturing pluripotent stem cells (e.g., iPSC) in a basal medium containing BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), activin A, and a ROCK inhibitor (e.g., Y-27632). In some aspects, the basal medium comprises about 10 ng / mL to about 50 ng / mL BMP4, about 10 ng / mL to about 100 ng / mL VEGF, about 0.5 μM to about 3 μM GSK3 inhibitor (e.g., CHIR99021), about 5 ng / mL to about 50 ng / mL activin A, and about 1 μM to about 20 μM ROCK inhibitor (e.g., Y-27632). In some aspects, the basal medium comprises about 30 ng / mL BMP4, about 50 ng / mL VEGF, about 1.5 μM GSK3 inhibitor (eg, CHIR99021), about 25 ng / mL Activin A, and about 10 μM ROCK inhibitor (eg, Y-27632).

[0078] In some respects, the differentiation method provided herein is included in culturing cells in a basal medium comprising VEGF and a TGFβ inhibitor (e.g., SB431542). In some respects, the basal medium comprises about 10 ng / mL to about 100 ng / mL VEGF and about 1 μM to about 20 μM TGFβ inhibitor (e.g., SB431542). In some respects, the basal medium comprises about 30 ng / mL VEGF and about 10 μM TGFβ inhibitor (e.g., SB431542).

[0079] In some respects, the differentiation method provided herein is included in culturing cells in the basal medium comprising TGFβ3 and PDGF (e.g., PDGF-BB). In some respects, the basal medium comprises about 1ng / mL to about 5ng / mL TGFβ3 and about 2ng / mL to about 8ng / mL PDGF (e.g., PDGF-BB). In some respects, the basal medium comprises about 2ng / mL TGFβ3 and about 4ng / mL PDGF (e.g., PDGF-BB).

[0080] In some aspects, the differentiation methods provided herein comprise:

[0081] (i) culturing pluripotent stem cells (e.g., iPSCs) in a basal medium comprising BMP4, VEGF, a GSK3 inhibitor, activin A, and a ROCK inhibitor;

[0082] (ii) culturing the cells in (i) in a basal medium comprising VEGF and a TGFβ inhibitor; and

[0083] (iii) culturing the cells in (ii) in a basal medium containing TGFβ3 and platelet-derived growth factor (PDGF) to form stromal cells or pericytes.

[0084] In some aspects, the method further comprises culturing the cells in (ii) in a basal medium before (iii). In some aspects, the method further comprises culturing the cells in (iii) in a basal medium (iv). In some aspects, the cells in (ii) are not passaged before (iii).

[0085] In some aspects, the differentiation methods provided herein include culturing pluripotent stem cells (e.g., iPSCs) in a basal medium comprising BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), and activin A. In some aspects, the basal medium comprises about 10 ng / mL to about 50 ng / mL BMP4, about 10 ng / mL to about 100 ng / mL VEGF, about 0.5 μM to about 3 μM GSK3 inhibitor (e.g., CHIR99021), and about 5 ng / mL to about 50 ng / mL activin A. In some aspects, the basal medium comprises about 30 ng / mL BMP4, about 50 ng / mL VEGF, about 1.5 μM of a GSK3 inhibitor (e.g., CHIR99021), and about 25 ng / mL activin A.

[0086] In some aspects, the differentiation methods provided herein comprise:

[0087] (i) culturing pluripotent stem cells (eg, iPSCs) in a basal medium comprising BMP4, VEGF, a GSK3 inhibitor (eg, CHIR99021) and activin A;

[0088] (ii) culturing the cells in (i) in a basal medium comprising VEGF and a TGFβ inhibitor (e.g., SB431542);

[0089] (iii) culturing the cells in (ii) in a basal medium; and

[0090] (iv) culturing the cells in (iii) in a basal medium comprising TGFβ3 and PDGF (eg, PDGF-BB) to form stromal cells or pericytes.

[0091] In some aspects, the method further comprises (v) culturing the cells in (iv) in a basal medium. In some aspects, the cells in (ii) are not passaged prior to (iii).

[0092] In some aspects of any of the methods disclosed herein, at the start of the culture of (i), the PSC (e.g., iPSC) is not fully confluent. In some aspects of any of the methods disclosed herein, at the start of the culture of (i), the PSC is at a confluence of about 50% to about 80%. In some aspects of any of the methods disclosed herein, at the start of the culture of (i), the PSC is at a confluence of about 50%, about 60%, about 70%, or about 80%.

[0093] In some aspects of any of the methods disclosed herein, the stromal cells are pericytes. III. Other aspects

[0094] The present disclosure also relates to stromal cells produced by any of the differentiation methods disclosed herein. The present disclosure also relates to pericytes produced by any of the differentiation methods disclosed herein.

[0095] The present disclosure also relates to an organoid comprising stromal cells and / or pericytes prepared by any differentiation method disclosed herein. As used herein, the term "organoid" refers to a differentiated or partially differentiated three-dimensional (3D) cell structure derived from pluripotent stem cells (e.g., iPSCs), which is self-organized by dense aggregation of cells in a controlled space. Such structures can be constructed to replicate most of the complexity of an organ, or to express specific aspects thereof, for example, to produce only certain types of cells.

[0096] Methods for maintaining differentiated stromal cells, pericytes, and organoids are well known and include culturing the cells or organoids in a cell culture medium as described herein and / or cryopreservation. Methods for preparing organoids generally include culturing cells in a 3D matrix. Suitable 3D matrices include, but are not limited to, polymers (natural or synthetic), ceramics, or composite materials. The 3D matrix can be in the form of a hydrogel, a porous 3D scaffold, a rapid prototyping scaffold, a foam, a sponge, a mesh, a microparticle, a fibrous network, a matrix mixture produced by a naturally occurring cell line (e.g., Matrigel TM ) and combinations thereof, such as microparticle-loaded hydrogels.

[0097] The present disclosure also relates to methods of promoting neovascularization or vascular development comprising administering stromal cells, pericytes, or organoids prepared by any of the differentiation methods disclosed herein.

[0098] The present disclosure also relates to methods of treating vasculitis or vascular disease comprising administering stromal cells, pericytes, or organoids prepared by any of the differentiation methods disclosed herein.

[0099] The present disclosure also relates to a method for treating cardiovascular disease, the method comprising administering stromal cells, pericytes or organoids prepared by any differentiation method disclosed herein. In some aspects, the cardiovascular disease is coronary artery disease (CAD), arrhythmia, heart failure, valvular heart disease, pericardial disease, cardiomyopathy (myocardial disease) or congenital heart disease.

[0100] The present disclosure also relates to certain differentiation media.

[0101] In some aspects, the disclosure provides a differentiation medium comprising a basal medium, BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), activin A and a ROCK inhibitor (e.g., Y-27632). In some aspects, the differentiation medium comprises a basal medium, about 10 ng / mL to about 50 ng / mL BMP4, about 10 ng / mL to about 100 ng / mL VEGF, about 0.5 μM to about 3 μM GSK3 inhibitor (e.g., CHIR99021), about 5 ng / mL to about 50 ng / mL activin A and about 1 μM to about 20 μM ROCK inhibitor (e.g., Y-27632). In some aspects, the differentiation medium comprises a basal medium, about 30 ng / mL BMP4, about 50 ng / mL VEGF, about 1.5 μM GSK3 inhibitor (e.g., CHIR99021), about 25 ng / mL activin A and about 10 μM ROCK inhibitor (e.g., Y-27632).

[0102] In some aspects, the disclosure provides a differentiation medium comprising a basal medium, VEGF, and a TGFβ inhibitor (e.g., SB431542). In some aspects, the differentiation medium comprises a basal medium, about 10 ng / mL to about 100 ng / mL VEGF, and about 1 μM to about 20 μM TGFβ inhibitor (e.g., SB431542). In some aspects, the differentiation medium comprises a basal medium, about 30 ng / mL VEGF, and about 10 μM TGFβ inhibitor (e.g., SB431542).

[0103] In some aspects, the disclosure provides a differentiation medium comprising a basal medium, TGFβ3 and PDGF (e.g., PDGF-BB). In some aspects, the differentiation medium comprises a basal medium, about 1 ng / mL to about 5 ng / mL TGFβ3 and about 2 ng / mL to about 8 ng / mL PDGF (e.g., PDGF-BB). In some aspects, the differentiation medium comprises a basal medium, about 2 ng / mL TGFβ3 and about 4 ng / mL PDGF (e.g., PDGF-BB).

[0104] In some aspects, the present disclosure provides a differentiation medium comprising a basal medium, BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), and activin A. In some aspects, the differentiation medium comprises a basal medium, about 10 ng / mL to about 50 ng / mL BMP4, about 10 ng / mL to about 100 ng / mL VEGF, about 0.5 μM to about 3 μM GSK3 inhibitor (e.g., CHIR99021), and about 5 ng / mL to about 50 ng / mL activin A. In some aspects, the differentiation medium comprises a basal medium, about 30 ng / mL BMP4, about 50 ng / mL VEGF, about 1.5 μM GSK3 inhibitor (e.g., CHIR99021), and about 25 ng / mL activin A. Example

[0105] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. Example 1 Differentiation of induced pluripotent stem cells into stromal cells

[0106] An experiment was performed to differentiate human induced pluripotent stem cells (iPSCs) (BJRiPS iPSCs provided by Jintang Du) into stromal cells using the following protocol.

[0107] Day -1 : One day before the experiment, Growth factor reduced (GFR) basement membrane matrix ( Catalog No. 354230) on ice to thaw overnight.

[0108] Day 0 : On the day of initial iPSC inoculation, 120 μL GFR was added to 12 mL of Dulbecco's Modified Eagle's Medium (DMEM) / F12 medium (Thermo Fisher, product number 11320033) and mixed. 2 mL of this mixture was added to each well of a six-well plate and incubated at room temperature (RT) for at least 1 hour.

[0109] Use a cell culture plate containing iPSCs at approximately 70% confluence for initial seeding. Remove the culture medium from the plate and wash the iPSCs once with phosphate buffered saline (PBS) (Gibco, product number 12604-013). Then, add 1.5 mL of (Biolegend, product number 423201) and incubated at 37°C for 7 minutes. The cells were lysed and passed through a strainer to collect the cell clumps and introduced into the mTESR TM Culture medium (STEMCELL TM Technologies Inc.) to quench The MoxiCyte live cell assay kit and Moxi Flow instrument were then used to The cell solution was counted.

[0110] The cells were then seeded at 200,000 cells per well in a 10 μM Y-27632 (STEMCELL TM Technologies Inc.) TM culture medium and incubate overnight.

[0111] Day 1 : Remove medium from plates with unattached iPSCs and add 2 mL of fresh warm mTESR with 10 μM Y-27632 to each well of cells. TM Culture medium. Use The cells were imaged in their entire wells using a live cell analysis system (Sartorius). Figure 1A middle.

[0112] Day 2 : Remove medium from plates with unattached iPSCs and add 2 mL of fresh warm mTESR with 10 μM Y-27632 to each well of cells. TMCulture medium. Use The live cell analysis system images cells in their entire well. Example images are shown in Figure 1B middle.

[0113] Day 3 : Mesoderm differentiation. Remove medium from nearly fully confluent culture plates and replace with 2 mL of filtered mesoderm induction medium per well, containing APEL with 25 ng / mL Activin A (Peprotech, product number AF-120-14E), 30 ng / mL bone morphogenetic protein 4 (BMP4) (Peprotech, product number AF-120-05ET), 50 ng / mL vascular endothelial growth factor (VEGF), 1.5 μM CHIR99021 (Biovision, product number 1748-5), and 10 μM Y-27632. TM 2 Culture medium (STEMCELL TM Technologies Inc.). The cells were imaged whole-well using the Live Cell Analysis System and incubated for approximately 4 days. An exemplary image from day 4 is shown in Figure 1C and an exemplary image on day 5 is shown in Figure 1D middle.

[0114] Day 7 : Vascular differentiation. Remove the culture medium from the culture plate and replace with 2 mL per well of filtered vascular induction medium containing APEL with 50 ng / mL VEGF and 10 μM SB431542 (Tocris, product number 3211). TM 2. Culture medium. Use Cells were imaged whole-well using a live cell analysis system and incubated for approximately 4 days with one replacement of the vascular differentiation medium. Exemplary images of days 7, 9, and 10 are shown in Figure 1E , Figure 1F and Figure 1G .

[0115] Day 11 : Cell expansion. Harvest cells by removing the culture medium, wash once with PBS, and add 1.5 mL The cells were detached and then incubated at 37°C for 7 minutes. TM 2. Medium Quenching The cells were then filtered through a filter to remove cell clumps. Count the cells and then add them to 15 mL complete EGM. TM -2 medium (EGM TM-2 Endothelial Cell Growth Medium -2 BulletKit TM Cells were plated at 800,000 cells per plate in gelatin-coated T-75 flasks (Sigma, product number G1394; Falcon, product number 353136) in 1% EDTA (Lonza, product number CC-3156 and CC-4176). Cells were grown to approximately 90% confluence and fresh complete EGM was used every 3 days. TM -2 medium was replaced with culture medium. An exemplary image on day 17 is shown in Figure 1H middle.

[0116] Stromal / pericyte differentiation : By removing EGM TM -2 medium and 15 mL of pericyte growth medium (PromoCell) were added to start differentiation, and the pericyte growth medium had 2 ng / mL transforming growth factor-β3 (TGF-β3 or TGFβ3) (Peprotech, product number 100-36E) and 4 ng / mL platelet-derived growth factor-BB (PDGF-BB) (Peprotech, product number 100-14B). The cells were incubated for 3 days. An exemplary image of the resulting test differentiated cells is shown in Fig. 1I and Figure 1J middle.

[0117] Next, the test differentiated cells were validated by measuring the expression levels of various stromal markers and control markers using flow cytometry. An overview of the results of this validation showing relative expression levels is shown in Table 1. Results for the test differentiated cells are shown, as well as control endothelial cells, pericytes, fibroblasts, and smooth muscle cells. Table 1: Flow cytometry quality control indicators

[0118] Next, the test differentiated cells were further characterized by tube formation assay using the following protocol. The test differentiated cells were co-cultured with human pulmonary artery endothelial cells (HPAEC) with or without 1 μM imatinib mesylate (to inhibit angiogenesis) and tube formation was observed. Specifically, 30 μL of GFR coated the wells of a 96-well plate (Costar, product number 387). The plate was spun at 1500 revolutions per minute (RPM) for 1 minute and incubated at 30°C for 30 minutes. The cells were then detached from the culture plate, counted, and resuspended in (DMEM) / F12 medium to obtain 30 μL of medium per well. Each well received 30,000 human pulmonary artery endothelial cells (HPAEC) and 5,000 differentiated cells.

[0119] HPAECs were labeled green using Nuclight Green Lentivirus (Essen / Sartorius #4475). 100 μL of transfection reagent was added to 5 mL of culture medium and mixed. In another 5 mL of culture medium, 10 μL of the desired lentivirus was added and mixed. The two 5 mL preparations were then added together, mixed and added to a plate of approximately 70% confluence of HPAECs or pericytes. The plates were grown overnight in an incubator.

[0120] The culture medium and lentiviral preparation were removed and replaced with fresh culture medium and the plates were incubated overnight. The plates were incubated for one week with 1 μg of puromycin in the culture medium during daily culture medium changes.

[0121] Differentiated cells were stained with 0.35 μM Nuclight Rapid Red (Essen Bioscience) in PBS at 37°C for 20 min. The dye was then quenched with culture medium and the cells were dispensed. For the tube formation assay, after the gel, 30 μL of the cell suspension was carefully placed on top of it. The plate was placed The live cell analysis system was used and images were taken every hour.

[0122] like Figure 2 As shown, the test differentiated cells developed tube formation and colocalized with HPAEC, whereas control cells treated with imatinib mesylate did not form tubes.

[0123] Figures 3A-3C shows that at the three-hour time point, HPAEC co-cultured with the test differentiated cells ( Figure 3A ), HPAEC co-cultured with test differentiated cells treated with imatinib mesylate ( Figure 3B ) and co-culture of HPAEC and placental pericytes ( Figure 3C ). These results indicate that the test differentiated cells associate with HPAEC tubes in a similar manner as primary pericytes.

[0124] Furthermore, the tested differentiated cells grown longest in the expansion medium had the best properties based on flow cytometry results as well as stability of tube formation and duration of structure.

[0125] All publications, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was expressly and individually indicated to be incorporated herein by reference. In addition, the citation or identification of any reference in this application should not be construed as an admission that such reference constitutes prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. A method for differentiating pluripotent stem cells into stromal cells, comprising: (i) culturing pluripotent stem cells in a basal medium containing bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), a glycogen synthase kinase 3 (GSK3) inhibitor, activin A, and a Rho-associated coiled-coil kinase (ROCK) inhibitor; (ii) culturing the cells in (i) in a basal medium comprising VEGF and a transforming growth factor β (TGFβ) inhibitor; and (iii) culturing the cells in (ii) in a basal medium containing TGFβ3 and platelet-derived growth factor (PDGF) to form stromal cells.

2. The method of claim 1, further comprising: Prior to (iii), the cells in (ii) are cultured in basal medium.

3. The method according to claim 1 or 2, further comprising: (iv) culturing the cells in (iii) in basal medium.

4. The method of any one of claims 1 to 3, wherein the cells in (ii) are not passaged prior to (iii).

5. A method for differentiating pluripotent stem cells into stromal cells, comprising: (i) culturing pluripotent stem cells in a basal medium comprising BMP4, VEGF, a GSK3 inhibitor and activin A; (ii) culturing the cells in (i) in a basal medium comprising VEGF and a TGFβ inhibitor; (iii) culturing the cells in (ii) in a basal medium; (iv) culturing the cells in (iii) in a basal medium containing TGFβ3 and PDGF to form stromal cells.

6. The method of claim 5, further comprising: (v) Cultivating the cells in (iv) in basal medium.

7. The method of claim 5 or 6, wherein the cells in (ii) are not passaged prior to (iii).

8. The method of any one of claims 5-7, wherein the culture medium in (i) further comprises a ROCK inhibitor.

9. The method of any one of claims 1-8, wherein the ROCK inhibitor is Y-27632.

10. The method of claim 9, wherein Y-27632 is present in the culture medium at a concentration of about 10 μM.

11. The method of any one of claims 1-10, wherein the GSK3 inhibitor is CHIR99021.

12. The method of claim 11, wherein CHIR99021 is present in the culture medium at a concentration of about 1.5 μM.

13. The method of any one of claims 1-12, wherein BMP4 is present in the culture medium at a concentration of about 30 μg / mL.

14. The method of any one of claims 1-13, wherein VEGF is present in the culture medium at a concentration of about 50 ng / mL.

15. The method of any one of claims 1-14, wherein activin A is present in the culture medium at a concentration of about 25 ng / mL.

16. The method of any one of claims 1-15, wherein the TGFβ inhibitor is SB431542.

17. The method of claim 16, wherein SB431542 is present in the culture medium at a concentration of about 10 μM.

18. The method of any one of claims 1-17, wherein TGFβ3 is present in the culture medium at a concentration of about 2 ng / mL.

19. The method of any one of claims 1-18, wherein the PDGF is PDGF-BB.

20. The method of claim 19, wherein PDGF-BB is present in the culture medium at a concentration of about 4 ng / mL.

21. The method of any one of claims 1-20, wherein the stromal cells are pericytes.

22. The method of any one of claims 1-21, wherein the pluripotent stem cells are not fully confluent when the culturing of (i) is initiated.

23. The method of any one of claims 1-22, wherein at the time of initiating the culturing of (i), the pluripotent stem cells are at a confluence of about 50% to about 80%.

24. The method of any one of claims 1-23, wherein the pluripotent stem cell is an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), an embryonic germ cell, or an adult stem cell.

25. Stromal cells obtained by the method of any one of claims 1-24.

26. Pericytes prepared by the method of any one of claims 1-24.

27. An organoid comprising the stromal cell of claim 25.

28. An organoid comprising the pericyte of claim 26.

29. A differentiation medium comprising a basal medium, BMP4, VEGF, a GSK3 inhibitor, activin A and a ROCK inhibitor.

30. A differentiation medium comprising a basal medium, about 30 ng / mL BMP4, about 50 ng / mL VEGF, about 1.5 μM CHIR99021, about 25 ng / mL Activin A, and about 10 μM Y-27632.

Images